Tritiated β-endorphin

ABSTRACT

Tritiated β-endorphin is prepared by catalytic tritiation of a [3,5-diido-Tyr]-β-endorphin which in turn is readily obtained by solid phase synthesis. The tritiated β-endorphin preferably [3,5-diiodo-Tyr 27  ]-β-endorphin may be utilized as a radio-labelled tracer compound in radioimmunoassay for β-endorphin and also exhibits biological activity comparable to that of the native β-endorphin.

BACKGROUND OF THE INVENTION

An immunoassay for β-endorphin including the use of various radiolabelled β-endorphin tracer compounds is described in U.S. Pat. No. 4,096,237, issued June 20, 1978, inventor, C. H. Li. Tritiated β-endorphin is disclosed in such patent.

The synthesis of N.sup.α -Boc-3,5-diiodotyrosine-(OBrBzl) is described by Lemaire et al., J. Am. Chem. Soc. 99, 1577 (1977).

Tritiation of a 3,5-diiodotyrosine ACTH analog is disclosed by Ramachandran and Behrens, Biochem. Biophys. Acta 496, 321 (1977).

DESCRIPTION OF THE INVENTION

The present invention relates to the preparation of tritiated β-endorphin, particularly tritiated human β-endorphin, by catalytic tritiation of an iodinated tyrosine containing β-endorphin analog. Such tritiation is conveniently carried out using palladium oxide as catalyst in a manner known per se. Note Ramachandran and Behrens,supra.

Suitable iodinated tyrosine-β-endorphin analogs useful as substrates for tritiation herein include [3,5-I₂ -Tyr¹ ]-, [3,5-I₂ -Tyr²⁷ ]- and [3,5-I₂ -Tyr¹,27 ]-β-endorphin.

EXAMPLE 1

[3,5-I₂ -Tyr²⁷ ]-β-endorphin was utilized by the solid-phase method as described in Example 6 of U.S. Pat. No. 4,038,222 with the exception that N.sup.α Boc-O-BrBzl-3,5-I₂ -Tyr is substituted for the corresponding tyrosine moiety in position 27 and chloromethylated styrene-divinylbenzene polymer is used. The iodinated peptide (6 mg, 1.5 μmole) was dissolved in 305 drops of 0.1 N HOAc, followed by 2.0 ml each of freshly distilled HMPA and DMA. To remove most of the water and HOAC from the reaction solution, it was evacuated at 21° C. to 0.05 mm Hg and kept at this pressure for approximately 45-60 min. This resulted in an approximate one-third reduction in volume and removed greater than 90% of the water as shown by NMR spectroscopy. The reaction solution could be kept frozen indefinitely at -70° before use. The catalyst used was PdO, finely ground to a dust in a glass mortar and pestle. An equal weight of catalyst to peptide was used with a tritium pressure of 700°-750° mm Hg°. The solution was magnetically stirred at 21° C. for 2.5-3.0 h. After removal of the excess tritium by evacuation, 50 mg of DTT in 0.5 ml of DMA was added. The solvent was then evaporated overnight at 21° C. to yield a brown-black residue. The residue was dissolved in 1-2 ml of a solution consisting of 5% HOAc AND 3% ethanol and submitted to a Sephadex G-10 column (20×400 mm) for desalting. Immediately after lyophilization of the peptide peak, it was chromatographed in a CMC column (10×500 mm) as previously described (Li et al., 1976), except that all buffers contained 3% ethanol (Evans, 1976). The contents in the major peak were lyophilized and submitted to partition chromatography on Sephadex G-50 in a 1.0×34 cm column in a solvent system consisting of butanol/pyridine/HOAc/H₂ O (5/0.04/1/4, v/v). The main component with R_(f) of 0.31 was lyophilized and rechromatographed on CMC as above to give a symmetrical peak which contains the tritiated β_(h) -endorphin. The content in the peak tubes could be stored indefinitely at 4° C. The yield of triated β_(h) -endorphin was 1.4 mg with 50 Ci/mmole.

The amino analyses of acid hydrolysates of native and tritiated β_(h) -endorphin showed that their compositions were identical and completely in accordance with that expected. Paper electrophoresis at pH 6.7, followed by autoradiography, showed a single spot for native β_(h) -endorphin (ninhydrin) and for the tritiated peptide (radioactivity) with identical mobility. Paper chromatography in the solvent system, n-BuOH/HOAc/H₂ O (4/1/5, v/v) which readily separates native and iodinated β_(h) -endorphin, showed no trace of iodinated peptide. The tryptic map of tritiated β_(h) -endorphin gave rise to three radioactive spots corresponding to peptide fragments of Asn-Ala-Tyr-Lys, AspAla-Tyr-Lys, and Asx-Ala-Tyr-Lys-Lys, respectively.

The biological activity of tritiated β_(h) -endorphin was identical to the native peptide as assayed in preparations of guinea pig ileum [Kosterlitz, Brit. J. Pharm. 39, 398 (1970)] with IC₅₀ of 0.70×10⁻⁸ M.

EXAMPLE 2

In analogy to the procedure of Example 1, [3,5-I₂ -Tyr²⁷ ] β_(c) -endorphin, synthesized in accordance to the procedure of Example 3 of U.S. Pat. No. 4,038,222 using N.sup.α -Boc-3,5-I₂ -Tyr(OBrBzl) in position 27 is tritiated to produce tritiated β_(c) -endorphin.

EXAMPLE 3

In analogy to Example 1,[3,5-I₂ -Tyr²⁷ ]-β_(p) -endorphin, synthesized in accordance to the procedure of Example 5 of U.S. Pat. No. 4,038,222 utilizing N.sup.α -Boc-3,5-I₂ -Tyr(OBrBzl) in position 27 of β_(p) -endorphin is tritiated to yield tritiated β_(p) -endorphin.

EXAMPLE 4

In analogy to Example 1, [3,5-I₂ -Tyr¹ ]-β_(h) -endorphin synthesized using N.sup.α -Boc-3,5-I₂ -Tyr(OBrBzl) to replace tyrosine in position 1 of β_(h) -endorphin is tritiated to yield tritiated β_(h) -endorphin.

EXAMPLE 5

In analogy to Example 1, [3,5-I₂ -Tyr¹,27 ]-β_(h) -endorphin, synthesized using N.sup.α -Boc-3,5-I₂ -Tyr(OBrBzl) to replace tyrosine in positions 1 and 27 of β_(h) -endorphin is tritiated to yield tritiated β_(h) -endorphin. 

I claim:
 1. Tritiated β-endorphin.
 2. The tritiated compound of claim 1 which is β-human endorphin.
 3. The tritiated compound of claim 1 which is β-porcine endorphin.
 4. The tritiated compound of claim 1 which is β-camel endorphin. 